Summary of work: Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress. These lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes and are able to examine the repair of different types of lesions and to measure different steps of the pathway. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. We have established in vitro assays using nuclear extracts from cells to measure the process of base excision repair of oxidative DNA damage. The experiments show that there are two forms of BER, the short and long patch, distinguishable by the length of the newly incorporated bases in DNA. We have shown that DNA polymerase beta is involved in both of these processes, and that it plays a critical role, possibly in the interaction with the flap-endonuclease FEN-1. We have disclosed a number of other interactions between proteins involved in the base excision repair pathway. We have reconstituted this process in vitro and are exploring both physical and functional protein interactions. This suggests that a major base excision repair complex exists which includes AP endonuclease, proliferating cell nuclear antigen (PCNA), polymerase (s), glycosylase(s) and others. These protein interactions are physical and functional and together support the "passing of buton" model, in which base excision repair takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. We are finding new protein partners in this process, including poly(ADP) ribose polymerase, which is activated by DNA starnd breaks. We are further examining these inetractions in a human disorder, Cockayne syndrome (CS), characterized by premature aging. There are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA, and this may be the major underlying cause of the disease. One base lesion, 8-oxo G, is of special interest since it causes mutations, if left unrepaired. We have studied the mechanism of repair of this lesion and find that it is repaired mainly via BER and in a mode that is not coupled to transcription.